Animals were used in accordance with the European Communities Council Directive 2010/63/EU. All efforts were made to minimize suffering and reduce the number of animals included in the study. All procedures were approved by the ethical committee for animal research of the University Paris Descartes (CEEA.34).

Surgical preparation and postoperative care for head implant surgery have been described previously (21, 22). Gas anesthesia was induced using isoflurane. The head was shaved using an electric razor. Lidocaine hydrochloride (2%; 2 mg/Kg) was injected locally before a longitudinal incision of about 2 cm was performed into the skin to expose the skull. A small custom-built head holder (3 × 3 × 5 mm) was fixed using dental cement (C&B Metabond; Parkellinc, Edgewood, NY, United States) to the skull just anterior to the lambda landmark. Following the surgery, animals were isolated and closely surveyed for 48 h. Buprenorphine (0.05 mg/kg) was provided for postoperative analgesia and care was taken to avoid hypothermia and dehydration.

The vestibulo-ocular pathway works as an open-loop: the vestibular signals trigger compensatory eye movements to stabilize gaze in the absence of sensory feedback. As a consequence, any imbalance or modification in the vestibular inputs leads to alteration of the eye movements triggered by head movements. This makes video-oculography the main tool used in hospitals to measure vestibular function. Eye movements were therefore used as a proxy to evaluate the efficacy of the vestibular system by quantification of the vestibulo-ocular reflexes of the mice.

To measure the neuronal effects of scopolamine, 220 μm-thick coronal brainstem slices were obtained from 5-week-old male C57BL/6J mice (n = 18) (24, 31). A total of 51 medial vestibular nuclei neurons (MVNn) were recorded with patch-clamp electrodes. The artificial cerebrospinal fluid (aCSF) used during the dissection and slicing is composed of (in mM): NaCl (120), NaHCO₃ (25), NaH₂PO₄ (1), KCl (2.5), MgCl₂ (3), CaCl₂ (0), glucose (10), sucrose (240). The recording solution differs only for NaCl (120), MgCl₂ (2), CaCl₂ (1) and sucrose (0). Analysis of resting discharge parameters, spike shape and classification of type A vs. type B neurons are similar to those previously reported (31). The intrinsic properties, as well as the responses to hyperpolarizing and depolarizing steps were compared between control conditions, or during pharmacological testing by the addition of muscarine (10 μM), or addition of muscarine (10 μM) + scopolamine (10 μM) to the bath. All chemicals were purchased from Sigma-Aldrich.

All mice were first tested during the Control protocol (Figure 1), then during one of the two counter-measure protocols (scopolamine, Figure 2, or visuo-vestibular mismatch, Figure 3). This approach allowed performing statistical analyses based on within-subjects models to account for non-specific and inter-individual variations. Since not all parameters were normally distributed (as tested with a Lilliefors test), we used the same non-parametric paired-test (Wilcoxon signed-rank test) to evaluate statistical significance in all conditions. When appropriate, a one-tail version was used to account for prior knowledge about the alternative hypothesis [e.g., the effect of a treatment on the parameters as the expected reduction of the VOR gain by the VVM protocol, Carcaud et al. (24)]. The thresholds for the statistical tests were adjusted using the Holm-Bonferroni method to account for the numerosity of the planed multiple comparisons. Although adjusted p-value thresholds were used to define the level of significance of the statistical tests, for ease of reading we report in the results section the corresponding uncorrected value noted with the ¢ symbol. All results in both the text and the figures are reported as mean ± standard deviation.

Scopolamine is known to help preventing motion sickness in humans. To determine if scopolamine has a comparable effect on mice, the effect of an injection of scopolamine on VOR and motion sickness were investigated on a subset of the animals (n = 16).

First, the effect of scopolamine on the Pica behavior was measured. Injection of scopolamine did not significantly change the baseline of the KIR in Sham condition (Table 2b, Figure 2A black vs. red bar; p > 0.1^¢). Then, the protective effects were tested by rotating the scopolamine-injected mice. While the rotation was efficient in provoking motion sickness in the absence of scopolamine (higher KIR with the MS protocol; Table 2a, Figure 2A, white bar; p < 0.05^¢), the KIR remained low when scopolamine was preemptively administrated (Table 2c, Figure 2A, gray bar). The KIR of scopolamine-injected mice after rotation was not different from that of the Control condition (no scopolamine; no rotation; Table 2d, Figure 2A black vs. gray bars; p > 0.1^¢). Thus, while rotation provoked the Pica behavior in these mice, preemptive injection of scopolamine protected them against MS.

To determine the interplay between motion sickness and vestibular sensitivity, the VOR and MOR of mice injected with scopolamine was recorded. Following the injection of scopolamine, but in the absence of rotation (no MS induction), the angular VOR gain of mice was decreased (p < 0.001^¢ at all tested frequencies; Figure 2B left panel). The decrease was in range ~60% at 0.2 Hz and ~40% at 0.5 and 1 Hz. Modifications of aVOR also affected the timing of the response with a tendency toward greater phase lead at 0.2 Hz (Δphase = +9°, p < 0.10^¢) and 0.5 Hz (Δphase = +15°, p < 0.01^¢). Notably, this effect of scopolamine was consistently observed in all injected mice tested before provocative rotation. Similarly, the MOR of scopolamine-injected mice was significantly reduced in both CW and CCW direction (Figure 2B, right panel; p < 0.01^¢ for both directions).

Since motion sickness and scopolamine injection both induce reduction of the vestibular gain and increase in phase leads, we asked whether their combination would lead to a greater attenuation of vestibular reflexes. When scopolamine-injected animals were provocatively rotated, the gain of the aVOR was found to stay significantly lower compared to control conditions (p < 0.01^¢ at all frequencies; Figure 2B, left panel). However, there was no additional decrease between the scopolamine and scopolamine+rotation groups (Figure 2C, left panel; all frequencies >0.05^¢). A similar result was found for the MOR_β which was significantly decreased by the scopolamine injection (Figure 2B, right panel, p < 0.001^¢) but was not different in scopolamine-injected mice tested with or without rotation (Figure 2C, right panel). This result demonstrates that the preemptive modification of the vestibular reflexes by scopolamine injection has occluded the effects on the VOR normally observed following rotation (MS induction).

Since the scopolamine injected groups did not show any obvious sign of MS (see behavioral proxies, Figure 2A), we interpret that in saline-injected mice the reduction of the vestibular reflexes could be causally related to the occurrence of motion sickness following rotation. Since scopolamine-injected mice do not suffer from motion sickness, we hypothesize that the diminution of the vestibular sensitivity (by scopolamine injection in this case) could act as a protective mechanism against motion sickness.

To test this hypothesis, we took advantage of a new methodology based on a long-lasting (14 days) visuo-vestibular mismatch (VVM, see Methods) which leads to a significant decrease in the gain of the VOR (24). Another subset of the mice (n = 8) was initially tested in control conditions and exposed to the provocative protocol. Before VVM, these mice had normal KIR, which again significantly increased following MS induction (Table 2e; p < 0.05^¢). Following these initial tests, the animals were left unperturbed for 48 h, before to start the VVM protocol. This methodology consists in putting on the head of the mouse a device which creates a visuo-vestibular mismatch. For 2 weeks, the animals were left in their home-cage with the apparatus on the head [see Figure 3A and protocol in Carcaud et al. (24)].

How does the VVM protocol affect vestibulo-ocular reflexes? As expected, the VVM protocol significantly reduced the gain of the VOR compared to pre-VVM values at all tested frequencies by >50% (Figure 3B compare black and dark blue bars; p < 0.05^¢). We then compared the maculo-ocular responses of mice before and after the VVM protocol. The MOR responses of the mice post-VVM were also significantly reduced compared to pre-VVM condition by about 50% in both clockwise and counterclockwise directions (p < 0.05^¢ for both directions; Figure 3B right panel). This result demonstrates that the VOR reduction following the long-lasting visuo-vestibular mismatch already reported for the canal-dependent pathway similarly reduces the otolith-dependent pathways, possibly through central mechanisms [see discussion in Carcaud et al. (24)].

Could the reduction of vestibular sensitivity following the VVM protocol prevent motion sickness? As expected the KIR of these mice was increased by the rotations before VVM (Table 2e, Figure 3A, right panel). After the VVM, the KIR was not significantly different from control conditions (p > 0.1^¢; compare black and deep blue bars on Figure 3A). When VVM mice were rotated (light blue bar), their KIR remained low, tended to be smaller compared to that of Shams (p > 0.1^¢) and similar to the non-rotated condition (p > 0.1^¢). The KIR in the VVM conditions tended to remain smaller from that of rotated mice (p < 0.10^¢ for both non-rotated and rotated VVM conditions), suggesting a protective effect of the VVM against MS.

To prevent de-adaptation of the reflexes, no Sham condition were attempted after removal of the device. VOR and MOR of the VVM mice were thus recorded immediately after removing the device (Pre values), and again immediately after MS rotation (Post values; Figure 3A). The effects of the provocative rotation were evaluated by comparing the PRE and POST effects (Figure 3B dark vs. white bars). As previously described (Figures 1B,C), the provocative rotation induced a reduction of angular VOR and MOR in control condition seen as a significant reduction in most tested conditions (compare black and white bars). Following the VVM protocol, rotation did no longer affect the efficacy of the vestibular reflexes, so that VOR gains and MOR bias all remained low and non significantly different between the VVM and VVM + rotated conditions (Figure 3B, compare blue and light blue bars).

Overall, these results show that rotations trigger in control mice MS symptoms (KIR increase) and lead to a reduction of the aVOR gain with increased phase lead, and to a decrease of MOR bias. Scopolamine or visuo-vestibular mismatch protocols both reduce the efficacy of the reflexes and offer some protection against motion sickness symptoms.

To understand the cellular mechanisms of scopolamine, pharmacological experiments were then conducted on vestibular neurons recorded on brainstem slices.

Motion sickness is a disease associated with discomfort, and often mistaken with the emetic reflex. While the association of MS and emesis is common in humans, it was demonstrated that rodents actually lack the brainstem neurological components responsible for emesis (32). However, the illness-response behavior known as Pica was identified as an analogous to vomiting, observed both in response to intoxication (33) and to provocative vestibular stimuli (29). The Pica behavior has since the 90's extensively been used as an index of rat motion sickness [e.g., (34, 39–41)] and was later validated in mice (42, 43). In both species, the causal relation between an intact vestibular system and the Pica behavior following challenging rotational stimuli was demonstrated (29, 43). Here, we have shown that in mice, Pica behavior can serve as a reliable index of motion sickness induced by a double-rotation paradigm similar to the one originally used in rats (29). We note that other behavioral symptoms such as piloerection, tremble, and abnormal urination were also frequently observed, although not quantified here. The stimulation protocol used is particularly efficient in generating combined canalar and otolithic overstimulation. Because of the possibility to use genetically-engineered mice and to conduct molecular studies, rodent models have recently attracted the attention of many research groups. Wang et al. (44–46) have studied in rats the inter-individual differences and the implication of the vestibulo-thalamic pathway in the habituation to provocative rotation. They also demonstrated that otoconia-deficient mice (het) are less susceptible to vestibular MS, indicating the pivotal role of otolithic overstimulation in MS generation. Wang and colleagues (47) took advantage of the mouse model to study the genetic susceptibility to MS by generating MS-susceptible or MS-resistant mouse strains. This recent work suggests the implication of a new protein, the swiprosin-1, in the vestibular-dependent response to MS. Collectively these studies demonstrate that Pica behavior constitute a reliable index of MS and reveal the high potential for combined genetic, molecular and physiological approaches in rodent models of MS.

Many studies have investigated the correlation between VOR characteristics and the susceptibility of the subject to MS, in order to use the VOR as a predictive measurement of MS. Overall, contradictory results were reported regarding angular VOR and occurrence of MS (48, 49). Ventre-dominey and colleagues (50) reported that MS susceptibility co-occurs with decreasing time constant of the VOR and with the increasing eye velocity during otolith-specific stimulation (OVAR); however other studies contradicted this result (51) and rather suggested an implication of the velocity storage in the genesis of MS during OVAR. Recently, Clement and Reschke (52) reported a correlation between MS susceptibility and the phase lead of the VOR at low frequency, with no correlation with VOR gain. Overall, studies in humans suggest a closer relationship between MS and VOR dynamic properties (phase) rather than VOR sensitivity (gain). Notably, human studies were conducted in order to evoke some degree of discomfort, but experiments were stopped just before or as soon as the subject reached sickness (52), limiting the exposure to motion to typically few minutes, which differs from our protocol. We note also that the rotation protocol used here (combination of 2 opposite directions of rotation with sinusoidal variation in angular speed) is more challenging than the protocols used in humans. Our results suggest that a lasting provocative vestibular stimulation leading to the occurrence of MS drives a significant decrease in the gain of vestibulo-ocular reflexes associated with an increase in the phase lead. This decrease in gain similarly concerned the semi-circular canals (angular VOR) and the otoliths (Maculo-ocular reflex), compatible with the hypothesis of a central common mechanism. Reduction of motion sickness following habituation was associated with a decrease in the time constant of the velocity storage (51, 53–56) and there is evidence for angular VOR gain reduction correlated with MS reduction in expert subjects, as for instance in skaters (57) or in sailors (58). A link between a higher aVOR gain and an increase in phase lead was also suggested as an indication of higher seasickness susceptibility (48). Within this framework, the general decrease in gain and increase in phase lead in the vestibulo-ocular responses we report, putatively associated with a decrease in the general sensitivity of the vestibular system, might reduce the sensitivity to the conflicting sensory inputs, and thus putatively help preventing later occurrence of MS.

If interactions between the VOR main parameters and MS exist, then it might be possible to act on the reflexes to manipulate the susceptibility of the patients. Dai et al. (1) demonstrated in a group of MS-susceptible patients that a visuo-vestibular iterated training protocol could reduce MS sensitivity for several weeks following the habituation sessions. We took advantage of a long-lasting visuo-vestibular mismatch to induce a reduction in the vestibulo-ocular reflexes that again affected equally both canals- and otolith-based reflexes. We demonstrated (24) that this protocol leads to a reduction of the neural responses in the direct VOR pathway. The cellular mechanisms associated to this decrease were a reduction in the synaptic efficiency between the vestibular afferent and the central vestibular neurons and a decrease in the excitability of subpopulations of central vestibular neurons (24). In other term, the long-lasting visual perturbation reduced the brainstem sensitivity to vestibular inputs. Here, we show in mice that the visually-induced reduction in the vestibular reflexes offers a protection against MS. Our results suggest that this effect lasts for at least 3 days, although longer term effects are possible and would deserve dedicated experiments. Overall, our neural and behavioral evidence support the possibility of using visuo-vestibular protocols to habituate susceptible patients to MS induced by vestibular overstimulation or by visuo-vestibular sensory conflicts. For example, since myopic people who wear glasses (but not lenses) have lower angular VOR gains (59), it would be interesting to test whether they are less susceptible to MS than myopic people wearing lenses, and even less than hyperopic people corrected with glasses, whose angular VOR gain is enhanced because of their high positive lenses. Because changes in the efficacy of gaze stabilizing systems are often associated with oscillopsia (60, 61), it would be interesting to study if patients under anti-motion sickness treatments report greater oscillopsia during active head motions.

Scopolamine is well-known as being among the most efficient anti-MS drugs in humans. It is commonly used in particular during space flight as a counter-measure against space motion sickness. In a series of experiments performed on humans in the 80's, Pyykkö et al. (62–64) demonstrated that patches of scopolamine prevented motion sickness by reducing the vestibular and optokinetic gains and suggested that the drug acted on the integrative function of the central vestibular nuclei. More recently, Werts et al. (14) reported a reduction of the angular VOR and caloric response following intranasal administration of scopolamine. Scopolamine had a depressant action on the response of the semicircular canals, postulated to be a combination of peripheral and central effects while it had little effect on the saccular reflex tested with cervico Vestibular Evoked Myogenic Potentials (cVEMP). On the other hand, Tal et al. (65) reported a significant decrease in cVEMP p13 latency following scopolamine administration. Bestaven et al. (16) demonstrated a significant reduction of ~30% of the vestibulo-spinal reflexes following galvanic vestibular stimulation associated with a decrease in balance test and vertical perception. In cat, no direct effect of scopolamine on the VOR was found at low doses, while at high doses the effects were confounded by sedation (66). To our knowledge, our experiments for the first time demonstrate in rodents that the prophylactic effect of scopolamine is associated with a reduction of vestibular sensitivity that concerns not only the semi-circular canal but also the otolith signals. We further show that the preemptive reduction of the vestibular reflexes by scopolamine injection can occlude the reduction of the VOR normally observed following MS. This occlusion suggests that both phenomena rely on a single mechanism or that, if the two processes are distinct, they converge on the same neuronal elements that cannot be adapted below a certain threshold.

What are the neuronal mechanisms associated with MS? Experimental evidence suggests that the processing of divergent sensory inputs in various brain areas (e.g., cerebellum; thalamus) contributes to patients' MS and also impacts the functioning of many cortical areas (67, 68). A key observation which emphasizes the instrumental role of the vestibular system may be, however, that patients with a total loss of labyrinthine function do not get motion sick [review in Lackner (5)]. In addition, in most instances it is the exposure to passive, rather than active, motion that leads to MS (69). In the vestibular nuclei and in the fastigial nuclei of the cerebellum, neurons categorized as “vestibular-only” were demonstrated to differentially encode passive and active movements (70–74). The proposed mechanism is termed “reafference cancelation.” It suggests that the vestibulo-cerebellum is using an internal model to predict the consequences of active, voluntary movements and substract this reafference signal from the signal sensed by the vestibular organs, termed exafference signal. As a result of the substraction of reafference and exafference, the discharge of vestibular-only neurons would represent the difference between the expected movement and the actual movement. Their discharge thus codes the “unexpected,” passive part of head movements. Vestibular-only neurons are implicated in vestibulo-spinal and vestibulo-sympathic pathway and are nowadays the best candidate for motion sickness generation within the vestibulo-cerebellum (73).

The identification of vestibular-only neurons in vitro still remains to be done. However, recent data have suggested that type B neurons constitute the vestibular-projection neurons while type A neurons would constitute the interneurons implicated in local regulation of activity (24, 35). It was also demonstrated that VN neurons that project to the cerebellum and are implicated in vestibulo-cerebellar regulatory loops are glutamatergic, so there is a high probability that vestibular-only neurons and neurons that project on the cerebellum are dominantly type B neurons. Here, all tested type B neurons were found to be modulated by cholinergic stimulation. The presence of nicotinic and muscarinic acetylcholine receptors (mAChR) in the vestibular nucleus with high density in the medial vestibular nucleus is well documented (75–79). Two distinct populations of type B neurons were found based on their modulation by the cholinergic system. Acetylcholine had opposite effects on these subpopulations, suggesting the existence of different receptors. Zhu et al. (38) reported that among the five mAChR subtypes, M2 and M3 may be the most highly expressed in the rat MVN. Interestingly, M2 is linked to the excitatory Gq/11 proteins, while M3 is coupled to the inhibitory Gi/o proteins (80, 81), and both receptors could play distinct roles in regulating vestibular afferent activity onto MVN neurons and activity of cerebellum-projecting neurons (38). The potential of mACHR subtype-specific agonists and antagonists as counter-measures again MS should be the focus of future studies.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.